Digestive and Liver Disease - Volume 48 - Supplement 2 - February 2016

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e76 Abstracts of the 22nd National Congress of Digestive Diseases / Digestive and Liver Disease 48S2 (2016) e67–e231
determine if HNE degrades infliximab, rendering it ineffective, and
if elafin can prevent these effects.
Material and methods: Perendoscopic intestinal biopsies were
collected from the inflamed mucosa of 35 patients with UC,
29 patients with Crohn’s disease (CD) and 30 control subjects.
Elastinolytic activity was determined by elastase activity assay,
whereas elastin was assessed by van Gieson staining and ELISA.
Elafin expression was evaluated by ELISA, immunoblotting and
immunofluorescence. The effect of elafin on elastinolytic activity of
UC biopsies was investigated in vitro. After co-incubation with HNE,
the integrity and the TNF-alpha neutralizing function of infliximab
were studied by immunoblotting or using a nuclear factor-kB
reporter cell line, respectively.
Results: Mucosal samples from inflamed areas of UC patients
displayed significantly (p<0.05) higher elastinolytic activity and
reduced elastin expression (mean 17.3 ng/mg, SEM 2.8) compared to
CD (37.6 ng/mg, SEM 5.8) and control subjects (44.4 ng/mg, SEM 6.5).
Levels of elafin were paradoxically increased in UC in comparison to
control subjects and, to a lesser extent, to CD. However, addition of
elafin restored elastinolytic activity to normal levels. HNE degraded
infliximab and this cleavage was inhibited by elafin. Infliximab
largely lost its ability to neutralize TNF-alpha after co-incubation
with HNE.
Conclusions: The increased activity of HNE in UC may affect
therapeutic efficacy of infliximab agents in these patients, thus
providing an explanation for the unresponsiveness to infliximab in
UC patients.
OC.02.3
SMAD7 KNOCKDOWN RESTORES ARYL HYDROCARBON
RECEPTOR EXPRESSION AND PROTECTIVE SIGNALS IN
INFLAMMATORY BOWEL DISEASE
Monteleone I.1, Marafini I.1, Zorzi F.1, Di Fusco D.1, Bevivino G.* 1,
Rizzo A.1, Sileri P.2, Sica G.2, Pallone F.1, Monteleone G.1
1Cattedra di Gastroenterologia, dipartimento di Medicina dei Sistemi,
Università Tor Vergata, Rome, Italy, Roma, Italy, 2dipartimento di
Chirurgia, Università Tor Vergata, Roma, Italy
Background and aim: Excessive T cells mediated immune response
promotes pathogenic inflammation in the gut. Such abnormal T-cell
response is in part due to a defective activity of counter-regulatory
mechanisms. Aryl hydrocarbon receptor (AhR), a cytosolic
transcription factor known for mediating the toxicity of xenobiotic
molecules as dioxin, delivers protective and anti-inflammatory
signals in the gut. Expression of AhR is markedly reduced in Crohn’s
disease (CD), and this defect contributes to amplify inflammatory
signals. Since CD lamina propria (LP) T cells are resistant to TGF-
beta1-mediated immunesuppression due to high Smad7, an
inhibitor of TGF-beta1 activity, we examined whether the reduced
AhR expression in CD relies on high Smad7.
Material and methods: AhR and IL-22 were evaluated in normal
LPMC stimulated with TGF-β1 and 6-formylindolo[3,2-b]carbazole
(Ficz), a high-affinity ligand of AhR, and in CD LPMC incubated with
a specific antisense oligonucleotides for Smad7 and then stimulated
with Ficz in presence or absence of TGF-beta1. AhR expression was
also evaluated in LP T cells isolated from transgenic mice over-
expressing Smad7 in T cells and the Smad7-dependent down-
regulation of AhR was studied in the trinitrobenzene-sulfonic-acid
(TNBS) model of colitis.
Results: In normal LPMC, TGF-beta1 induced AhR and this event
associated with increased production of IL-22 following stimulation
with FICZ. Inhibition of Smad7 in CD LPMC restored TGF-b1
signaling and enabled TGF-b1 to boost AhR expression. Consistently,
AhR expression and consequently Ficz-mediated IL-22 production
were markedly reduced in T cells isolated from Smad7 transgenic
mice. Moreover, Smad7 transgenic mice injected with Ficz were not
protected against TNBS induced colitis.
Conclusions: Smad7 sustains the defective expression of AhR thus
contributing to amplify and maintain pathological process in the
gut.
OC.02.4
OXIDATIVE STRESS AND THROMBOXANE-DEPENDENT PLATELET
ACTIVATION IN INFLAMMATORY BOWEL DISEASE (IBD): EFFECTS
OF ANTI-TNF-ALFA TREATMENT
Di Sabatino A.*1, Santilli F.2, Guerci M.1, Giuffrida P.1, Aronico N. 1,
Simeone P.2, Caprotti A.1, Salvatore C.1, Tripaldi R.2, Liani R.2,
Ardizzone S. 3, Massari A.3, Balduini A. 4, Malara A.4, Corazza G.R.1,
Davì G.2
1 First Department of Medicine, IRCCS San Matteo Foundation, Pavia,
Italy, 2Internal Medicine and Center of Excellence on Aging, “G.
d’Annunzio”, Chieti-Pescara, Italy, 3Gastroenterology Department,
“L.Sacco” Hospital, Milan, Italy, 4Department of Molecular Medicine,
Laboratory of Biotechnology, IRCCS San Matteo Foundation, Pavia,
Italy
Background and aim: Patients with IBD, namely Crohn’s disease
(CD) and ulcerative colitis (UC), have a higher risk of coronary
artery disease (CAD) despite having a lower burden of traditional
risk factors. We previously reported that several inflammatory
diseases are associated with enhanced lipid peroxidation and
persistent platelet activation. Platelets from patients with CD
release more soluble CD40L than control subjects and this might be
responsible for the platelet hyperactivation observed in CD. Thus,
we tested the hypothesis that the urinary levels of F2-isoprostane
8-iso-prostaglandin (PG) 2 a, marker of oxidative stress, and urinary
11-dehydro-thromboxane (TX) B2 and plasma CD40L, markers of
platelet activation, are enhanced in IBD patients.
Material and methods: Urinary samples were taken from 129 IBD
patients (59 CD, 70 UC, mean age 45±15 yrs, 81 males) and 37 healthy
subjects (mean age 38±17 yrs, 8 males). Among CD patients, 51 had
ileo-colonic disease, and 8 had ileal disease. Among UC patients,
34 had a left-sided colitis and 36 had a pancolitis. Urinary levels
of 8-iso-PGF2a and 11-dehydro-TXB2, as well as plasma CD40L
levels, were measured in IBD patients and control subjects and a
cross-sectional comparison was performed. Among all subjects, 13
patients on chronic treatment with biologic agents were followed
up for up to 6 months.
Results: IBD patients had significantly higher urinary 8-iso-PGF2a
and 11-dehydro-TXB2 as well as plasma CD40L than control subjects.
Interestingly, those with the urinary excretion of both 8-iso-PGF2a
and 11-dehydro-TXB2 in excess of 2000 pg/mg creatinine had higher
disease activity scores. In both IBD patients and control subjects, a
significant direct correlation was found between urinary 8-iso-
PGF2a and 11-dehydro-TXB2 (Rho=0.639, p<0.0001). Plasma CD40L
was also significantly related to both 8-iso-PGF2a and 11-dehydro-
TXB2 (Rho=0.604, p<0.0001, and Rho=0.895 and p<0.0001,
respectively). These associations remained highly significant when
considering the whole group of patients, without controls, or
the single diseases (UC or CD). In particular, in UC patients, both
CD40L and 11-dehydro-TXB2 were also directly correlated with the
clinical activity index (CAI) (Rho=0.544, p<0.0001; Rho=0.665, p
<0.0001). Initiation of biologic agents in 13 patients was associated
with a significant reduction of the urinary excretion of both 8-iso-
PGF2a and 11-dehydro-TXB2 after 2 months (p=0.008 for both)
and 6 months (p=0.008 for both) of follow-up, concurrent with
improvement in clinical activity indices.
Conclusions: This study supports the presence of enhanced TX-
dependent platelet activation in IBD patients. Further studies are
underway in order to establish whether urinary and blood levels